Identificaton of the Alarm and Sex Pheromones of the Leaf?Footed Bug, Leptoglossus zonatus (Heteroptera: Coreidae)

Article abstract of DOI:10.21577/0103-5053.20180238

The leaf-footed bug, Leptoglossus zonatus (Heteroptera: Coreidae), is mainly found in maize fields in Brazil. In this work, we elucidated and quantified the volatile compounds from nymphs and adults of L. zonatus. The defensive compounds produced by nymphs are aldehydes and oxo-alkenals, with some compounds showing different proportions between instars. For the first time, 5-ethyl-2(5H)-furanone has been identified as a defensive compound for a species of this family. In adults, the alarm pheromone includes hexanal, 1-hexanol, hexanoic acid, hexyl acetate, and octyl acetate. Males and females produce the same compounds, however, hexanal showed a significant difference between sexes. Gas chromatography (GC) analysis of adult aeration extracts revealed male specific compounds, which attactiveness to females was confirmed through olfactometer bioassays. By using a GC-electroantennographic detector (EAD), it was shown that female antenna presented electrophysiological responses to five components ((Z)-β-ocimene, (Z,Z)-allo-ocimene, decanal, α-trans-bergamotene, and (E)-β-farnesene) from male aeration extracts. In this way, we propose that males are responsible for emission of a sex pheromone in this species.

Full text of DOI:10.21577/0103-5053.20180238

http://dx.doi.org/10.21577/0103-5053.20180238
J. Braz. Chem. Soc., Vol. 30, No. 5, 939-947, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Article
Identificaton of the Alarm and Sex Pheromones of the Leaf‑Footed Bug,
Leptoglossus zonatus (Heteroptera: Coreidae)
Katia M. Inoue,a Diogo M. Vidal,b Emir B. Saad,a Camila B. C. Martinsa and
Paulo H. G. Zarbin *,a
aLaboratório de Semioquímicos, Departamento de Química, Universidade Federal do Paraná (UFPR),
PO Box 19081, 81531-990 Curitiba-PR, Brazil
bDepartamento de Química, Universidade Federal de Minas Gerais (UFMG),
31270-901 Belo Horizonte-MG, Brazil
The leaf-footed bug, Leptoglossus zonatus (Heteroptera: Coreidae), is mainly found in maize
fields in Brazil. In this work, we elucidated and quantified the volatile compounds from nymphs
and adults of L. zonatus. The defensive compounds produced by nymphs are aldehydes and oxo-
alkenals, with some compounds showing different proportions between instars. For the first time,
5-ethyl-2(5H)-furanone has been identified as a defensive compound for a species of this family. In
adults, the alarm pheromone includes hexanal, 1-hexanol, hexanoic acid, hexyl acetate, and octyl
acetate. Males and females produce the same compounds, however, hexanal showed a significant
difference between sexes. Gas chromatography (GC) analysis of adult aeration extracts revealed
male specific compounds, which attactiveness to females was confirmed through olfactometer
bioassays. By using a GC-electroantennographic detector (EAD), it was shown that female antenna
presented electrophysiological responses to five components ((Z)-β-ocimene, (Z,Z)-allo-ocimene,
decanal, α-trans-bergamotene, and (E)-β-farnesene) from male aeration extracts. In this way, we
propose that males are responsible for emission of a sex pheromone in this species.
Keywords: alarm pheromone, sex pheromone, Coreidae, 5-ethyl-2(5H)-furanone
Introduction
eight carbon atoms are commonnly found in the MTG
and DAGs of coreids. The most common compounds
produced by these glands are α,β-unsaturated aldehydes
and oxo-aldehydes.5,6
The amount of defensive compounds produced by
adults is frequently larger than the attractant pheromones.
One way to avoid the suppression of attractant compounds
by the alarm pheromone is to collect the volatiles through
aeration of undisturbed insects,7 or collect volatiles from
individual insects.8
Studies have shown that males of Leptoglossus spp.
produce specific compounds, such as aromatic compounds
and sesquiterpenes.5,9 In Amblypelta lutescens lutescens
(Heteroptera: Coreidae), field assays suggested that
sesquiterpenes can be envolved as an aggregation
pheromone.10 However, studies that attempted to work
with attractants for coreids have shown attractiveness
only to natural volatiles and not to synthetic components
in field tests.5,11 In this way, research is still needed to
confirm the chemical components responsible for the
sexual attraction in Coreidae, since a better understanding
The leaf footed-bug, Leptoglossus zonatus Dallas
(1851) (Heteroptera: Coreidae), is a polyphagous pest that
feeds on maize, cotton, blackberry, mango, citrus, beans,
guava, peach, soybean and many other crops.1 It has been
reported in several countries in America, extending from
United States to southernArgentina.2 In Brazil, both coreid
nymphs and adults feed on corn cobs and leaves and can
infect plants with fungal yeast that can lead up to 15% of
total production loss.2,3
Adults and nymphs of Heteroptera are characterized
by the release of irritating volatiles, used as defense
against predators and also as alarm pheromones.4 In
coreid nymphs, the odoriferous compounds are produced
in the dorsal abdominal glands (DAGs). In some adults,
the DAGs are not present and the metathoracic scent
gland (MTG) produces the alarm pheromone instead.5
Mixtures of aldehydes, alchools and acetates of six and
*e-mail: pzarbin@ufpr.br

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Identificaton of the Alarm and Sex Pheromones of the Leaf-Footed Bug, Leptoglossus zonatus
J. Braz. Chem. Soc.
of sexual communication and behavior of Coreids can be
a useful tool for sustainable pest management.
varied between the insects stage. The exuviae used in the
extraction process were collected ≤ 24 h after the insect
ecdysis. Five extracts of ten 1st instar exuviae, five extracts
of eight 2nd instar, five extracts of four 3rd instar exuviae,
five extracts of two 4th instar exuviae, and five extracts of
one 5th instar exuviae were obtained, and stored at –20 °C
until analysis.
Leptoglossus spp. population control on crops can
be accomplished through organophosphate pesticides,12
compounds that are associated with the increase of cancer
risk, dementia and potentially involved with Parkinson’s
disease.11 However, there is little report in the literature
about enviromentally-friendly management methods for
these insects.12 There are studies that report the use of
parasitoids of L. zonatus eggs and adults, but with low
parasitism percentage in laboratory tests.3 As an alternative
control method, the sex pheromones are efficient, less
harmful to the environment, and relatively inexpensive.13
Sex pheromones are typically species-specific and do not
affect other insects, such as natural enemies.14 Due to the
high sensitivity of insects olfaction systems, pheromones
can be used at very low concentrations, mainly presenting
low toxicity to humans, being considered safe for
agricultural purposes.15
Extraction of metathoracic glands (MTGs) contents
Adults were killed by freezing and dissected immersed
in water. In a Petri dish, each insect was pinned in the
prothorax in a dorsal-side-up position and the wings were
removed. The lateral margins of the abdomen were cut
up to the metathorax, and the dorsal cuticle was removed
exposing the viscera that were carefully removed. The
glandular complex in the metathorax (composed of the
orange reservoir and the lateral accessory glands) was
removed, dried with a tissue paper and immersed in
1000 μL of double distilled high-performance liquid
chromatography (HPLC) grade hexane for 24 h. The
extracts were stored at –20 °C until analysis. Three MTG’s
extracts of each sex were analyzed by gas chromatography-
mass spectrometry (GC-MS).17
The present work aimed to analyze L. zonatus DAG
compounds that remains in the exuviae of the five nymphal
instars and the MTG chemical components of adults. We
also intended to investigate the chemical composition and
attractiveness of the sex pheromone of L. zonatus.
Experimental
Collection of volatiles
Insect rearing
One cohort of five males and one cohort of five females
were placed in separate glass chambers (25 × 9 cm i.d.)
containing slices of corn cob and a continuous 1 L min-1
flow of humidified and charcoal-filtered air. The volatiles
were collected in a glass trap (11 × 3 mm i.d.) containing
20 mg of HayeSep Q 80-100 mesh (Sigma-Aldrich,
Steinheim, Germany). Male and female glass traps were
eluted with 400 μL of double distilled HPLC grade hexane
to collect the volatiles released by each sex and stored at
–20 °C until analysis.17 Extracts from male and female were
collected every 24 h for comparison.
In April (2016 and 2017), adults and immatures
of L. zonatus were manually collected in maize fields
(Zea mays) from the Instituto Agronômico do Paraná
(IAPAR), in Londrina-PR, Brazil (23°21’S, 51°09’W).
The insects were transferred to the Laboratório de
Semioquímicos at the Universidade Federal do Paraná
(UFPR), where they were separated by stage (adults and
immatures) and kept at 25 2 °C, 65 15% relative
humidity (RH), 12:12 h light:dark. Insects were reared
in plastic cages (35 × 20 × 20 cm) and fed with corn cob
(Z. mays), green beans (Phaseolus vulgaris), peanuts
(Arachis hypogaea), glossy privet (Ligustrum lucidum) and
cherry tomatoes (Solanum lycopersicum var. cerasiforme)
with replacement every 3 days. Adults were reared in
laboratory for at least 15 days prior bioassays.
Chemical analysis
Volatile extracts were analyzed by GC-MS (Shimadzu
GC-QP 2010 Plus, Tokyo, Japan) with electron ionization
mass detector operating in the electron impact (EI) mode
(70 eV) with an RTX-5 capillary column (30 m × 0.25 mm
i.d. and 0.25 μm film thickness; Restek, Bellefonte,
PA, USA). Samples were analyzed in splitless mode
with injector temperature at 250 °C. The column oven
temperature was maintained at 40 °C for 1 min, raised to
250 °C at a rate of 7 °C min-1, and maintained at 250 °C
for 10 min with helium as carrier gas.
Extraction of dorsal abdominal glands (DAGs) contents
DAGs reservoirs shed with exuviae were collected from
the exuviae of the five instars nymphs after ecdysis.16 The
exuviae extracts were obtained by extraction with 500 μL of
hexane, and the number of exuviae used on each extraction

Vol. 30, No. 5, 2019
Inoue et al.
941
Infrared spectra were obtained by gas chromatography-
Fourier transform infrared spectroscopy (GC-FTIR) using
a Shimadzu GC-2010 gas chromatograph coupled to a
DiscovIR-GC infrared detector (4,000-850 cm-1, resolution
of 4 cm-1; Spectra Analysis, Marlborough, MA, USA).
The GC was equipped with an EC™-5 capillary column
(Alltech, 30 m × 0.25 mm × 0.25 μm film thickness) and
helium as the carrier gas. The column oven temperature
program was the same used in the GC-MS analysis.
Chiral analysis were performed in a β-DEX™ 325
capillary column (30 m × 0.25 mm × 0.25 μm i.d.; Supelco,
Bellefonte, PA, USA) using a Shimadzu GC-17A gas
chromatograph equipped with a flame ionization detector
(FID) operated in split mode (1:1). The column oven
temperature was maintained at 50 °C for 1 min, raised to
130 °C at a rate of 1.5 °C min-1, raised to 200 °C at a rate
of 10 °C min-1 and maintained at 200 °C for 10 min with
nitrogen as carrier gas.
Volatile extracts were analyzed in a gas chromatograph
(Shimadzu GC2010, Tokyo, Japan) coupled with an
electroantennographic detector (Syntech®, Hilversum,
Netherlands) (GC-EAD) equipped with a DB-5 capillary
column (30 m × 0.25 mm × 0.25 μm; J&W Scientific Inc.,
Palo Alto, USA), operated in split mode (1:1, 250 °C)
with nitrogen as the carrier gas. The temperature program
started at 50 °C (1 min) and subsequently increased at
7 °C min-1 until 270 °C (10 min). The antennae were fixed
between the stainless steel electrodes using conductive gel
(Signa gel, Parker Laboratories, Inc., Fairfield, NJ, USA).
The electroantennogram was recorded on the Syntech
GCEAD32 software (version 4.6).
was isolated from the essential oil of Matricaria recutita.19,20
The essential oils were fractionated on a silica gel column
(6 × 3.5 cm) eluted with hexane.
Microderivatization
Catalytic hydrogenation with palladium on charcoal (Pd/C)
Exuviae extract in hexane and ca. 0.5 mg of Pd/C (10%)
were added to a glass vial. A rubber balloon filled with
hydrogen was attached to the vial, and the reaction was
stirred for approximately 2 h. The resulting solution was
filtered and analyzed by GC-MS.
Synthesis of 5-ethyl-2(5H)-furanone
5-Ethyl-3-(phenylselanyl)dihydrofuran-2(3H)-one (II)
Lithium diisopropylamide (LDA) was prepared by adding
an n-butyllithium solution (5.56 mL, 10.9 mmol, 1.96 M
in hexane) to a mixture of diisopropylamine (1.18 mL,
8.39 mmol) and tetrahydrofuran (THF, 18 mL) at –78 °C.
This solution was added dropwise to a stirred solution
of γ-caprolactone (0.312 mL; 2.8 mmol) in THF (9 mL)
and stirred for 75 min at –78 °C. A solution of diphenyl
diselenide (2.36 g, 7.55 mmol) in THF (12 mL) containing
hexamethylphosphoramide (HMPA, 1.46 mL, 8.39 mmol)
was added dropwise to the reaction flask at –78 °C over a
period of 50 min. The resulting mixture was stirred during
30 min at –70 °C and during 30 min at –40 °C. The reaction
was quenched by the addition of HCl solution (0.2 mol L-1)
until complete neutralization. The reaction was then extracted
with ethyl acetate, washed with saturated aqueous solutions
of NaHCO3 and brine, dried with Na2SO4, and concentrated
by rotary evaporation under reduced pressure. The crude
product was purified by column chromatography over silica
gel, eluted with hexane and hexane/ethyl acetate (95:5) to
furnish 5-ethyl-3-(phenylseleno)dihydrofuran-2(3H)-one in
87% yield.21 IR (ZnSe) νmax / cm-1 3056, 2969, 1765, 1580,
1479, 1439, 1192; 1H NMR (200 MHz, CDCl3) d 0.83-0.97
(m, 3H), 1.97-1.41 (m, 3H), 2.28-2.77 (m, 1H), 3.93-4.09
(m, 1H), 4.22-4.39 (m, 1H), 7.29-7.34 (m, 3H), 7.61-7.68
(m, 2H); 13C NMR (50 MHz, CDCl3) d 175.9, 135.4, 129.3,
128.8, 127.1, 80.6, 37.4, 35.8, 28.2, 9.3; MS (EI, 70 eV):
m/z (%): 270 (M+, 85), 223 (3), 195 (13), 183 (19), 157 (48),
145 (26), 131 (7), 116 (100), 91 (21), 77 (85), 69 (88), 55
(52), 41 (73).
1H and 13C nuclear magnetic resonance (NMR) spectra
of the synthetic compounds were recorded on anARX-200
spectrometer (Bruker, Billerica, USA) on 200 and 50 MHz,
respectively, as CDCl3 solutions. Chemical shifts are
expressed in ppm relative to TMS.
Identification of chemical compounds
The contents of volatile extracts of L. zonatus were
identified using the mass spectra analysis and retention index
(RI) of each compound and co-injections of volatile extracts
with reference standards. (E)-2-Hexenal, (Z)-2-hexenal,
and (E)-2-octenal were purchased from Acros Organics
(Geel, Turnhout, Belgium). (E)-4-Oxo-2-hexenal was
donated by Dr K. Chauhan of the USDA-ARS (Beltsville,
MD, USA).18 3,7-Dimethyl-1,3,6-octatriene, 2,6-dimethyl-
2,4,6-octatriene, and decanal were purchased fromAldrich
Chemical Company (Milwaukee, Wisconsin, USA).
α-trans-Bergamotene was obtained through its isolation
from the essential oil of Copaifera sp. and (E)-β-farnesene
5-Ethyl-2(5H)-furanone (5)
Aqueous H2O2 (8 μL, 0.1 mmol, 35% m/m in water)
was added dropwise to II (8.5 mg, 0.03 mmol) in THF
(450 μL), over 5 min at 0 °C. The mixture was stirred
at room temperature for another 2 h. The reaction was

942
Identificaton of the Alarm and Sex Pheromones of the Leaf-Footed Bug, Leptoglossus zonatus
J. Braz. Chem. Soc.
quenched with a saturated solution of aqueous NaHCO3
(0.1 mL, extracted with ethyl ether and concentrated
in vacuo to afford 5-ethyl-2(5H)-furanone (5) in 57%
yield.21 IR (ZnSe) νmax / cm-1 3095, 2976, 1747, 1600, 1464,
1170, 1109, 1012, 962, 904; 1H NMR (200 MHz, CDCl3)
d 1.02 (t, 3H, J 7.4 Hz), 1.66-1.92 (m, 2H), 5.01 (m, 1H),
6.13 (dd, 1H, J 5.7, 2.0 Hz), 7.45 (dd, 1H, J 5.7, 1.5 Hz);
13C NMR (50 MHz, CDCl3) d 173.1, 155.9, 121.8, 84.3,
26.3, 9.0; MS (EI, 70 eV): m/z (%): 112 (M+, 7), 84 (15),
83 (100), 57 (12), 55 (35).
0.01 0.00 mg (14, decanal), 1.5 0.53 mg (15, α-trans-
bergamotene) and 0.04 0.03 mg (16, (E)-β-farnesene)
(equivalent to 2 males).
Statistical analysis
Chromatographic peak area from each compound
present in exuviae and MTG extracts were compared
using analysis of variance (ANOVA) and the means were
compared by Tukey’s test (5%) using the statistical software
Minitab 17.22 After this, peak area was transformed to
percentage to better visualization of relative amounts of
each compound present on extracts. The olfactometer
bioassay data was analyzed through binomial test using
the statistical program BioEstat 5.0.23
Olfactometer bioassays
The behavior of male and female L. zonatus in the
presence of the natural male sex pheromone was observed
in a Y-olfactometer tube (4 cm in diameter, 40 cm long,
20 cm long arms) operated at 2 L min-1 flow of humidified
and charcoal filtrated air. The bioassay was conducted during
the photophase at 25 2.0 °C and 65 15% RH. A piece of
filter paper of 2 cm2 loaded with 20 μL of extract from males
was placed at the extremity of one arm of the tube and the
solvent was placed as a control in the extremity of the other
arm. The solvent of the solutions loaded to the filter paper
was evaporated before using it in the bioassay. Twigs of the
host plant (glossy privet) and a piece of corn cob were used
as attractant in both treatments. One virgin adult (male or
female) was placed in the entrance of the basal tube of the
olfactometer and its behavior was observed for 8 min. Twelve
individual males and 12 individual females were used in the
bioassay and a choice was scored whenever the insect walked
towards one of the arms and passed by 5 cm of the division
of the basal tube, staying there for at least 2 min.
Results and Discussion
DAGs and MTG chemical composition
Chromatograms of the DAGs and MTG extracts
(Figures 1 and 2) showed different compounds released
by nymphs and adults of L. zonatus. Six compounds were
present in the DAGs extracts and identified as (E)-3-hexenal
(1) (RI: 806), (Z)-2-hexenal (2) (RI: 847), (E)-2-hexenal
(3) (RI: 853), (E)-4-oxo-2-hexenal (4) (RI: 956), 5-ethyl-
2(5H)-furanone (5) (RI: 1035), and (E)-2-octenal (6)
(RI: 1059). The peak eluting after 4 (Figure 1) refers to
5-ethyl-3H-furan-2-one, known as a degradation product
from (E)-4-oxo-2-hexenal (4), due to extraction conditions.8
All compounds were identified through analysis of mass
and infrared spectra, comparisons of the retention indexes
and co-injection with synthetic standards.
The mean amounts of the EAD active compounds
present in the natural extract used in the bioassays were
0.35 0.02 mg (12, 3,7-dimethyl-1,3,6-octatriene),
0.13 0.06 mg (13, 2,6-dimethyl-2,4,6-octatriene),
The major components of the volatiles of nymphs are
(E)-4-oxo-2-hexenal (4) and (E)-2-hexenal (3) (Table 1).
On the first instar, the production of these compounds
Figure 1. Comparison between the exuvial extracts of Leptoglossus zonatus of the 4th instar and MTG extracts of adults.

Vol. 30, No. 5, 2019
Inoue et al.
943
data. Compound 5 was purified through fractionation of the
extract followed by a catalytic hydrogenation derivatization,
revealing one double bond since there was an increase of
two mass units on the derivative MS spectrum. The IR
spectrum of 5 (Figure 3b) showed a strong C=O stretching
band at 1746 cm-1 characteristic of an α,β-unsaturated
lactone. The mass spectrum (Figure 3d) showed a molecular
ion (M+) at m/z 112 and a base peak at m/z 83 due to the
loss of the side chain (C2H5+).
To confirm its structure, the lactone 5 was synthesized
and obtained from a mixture of stereoisomers as showed in
Scheme 1. The selenylation reaction of the γ-caprolactone I
with diphenyl diselenide resulted on II in 87% yield.
Compound 5 was obtained through selenoxyde elimination
from II, with H2O2, in 57% yield. The structure of 5 was
confirmed after co-injection of the natural extract and the
synthetic lactone.
Enantioselective gas chromatography was used to
determine the absolute configuration of the lactone through
comparison of the natural extract, the racemic mixture and an
enantiopure synthetic standard (Figure S7, Supplementary
Information (SI) section) using β-cyclodextrin based chiral
capillary column (β-DEX™ 325, Sigma-Aldrich, St. Louis,
USA). Synthetic racemic 5 was resolved and retention
times compared with (S)-5-ethyl-2(5H)-furanone (S-5),
obtained when (S)-γ-caprolactone was used as starting
material and with the natural compound, showing that it
occurs as a racemate.
The lactone 5 is commonly found in plant volatiles such
as tomato, soybean and guava.27,28 It was also identified
as a defensive compound in species of Pentatomidae
(Graphosoma lineatum and Halyomorpha halys), a
specie of Blattidae (Eurycotis floridana) and species of
Pyrrhocoridae.29,30
MTG extracts from adults showed five compounds
which were identified through retention indexes (RI)
and mass spectra: hexanal (7, RI: 807), 1-hexanol (8,
RI: 865), hexanoic acid (9, RI: 989), hexyl acetate (10,
RI: 1013) and octyl acetate (11, RI: 1210). MS spectrum
Figure 2. Leptoglossus zonatus defensive compounds identified in the
DAGs and alarm pheromone in the MTG.
reaches more than 98% of the whole extract, with 4 as
major component. However, 3 is produced in highest
amount by insects of the second to fifth instar, followed by
4. These compounds are commonly found in other coreid
species and in some other families of Heteroptera.5,6 They
can induce a dispersive behavior between conspecifics,
and paralyze or deter many predators, such as mantids.4,24
In Thasus neocalifornicus (Coreidae), the presence of
Wolbachia microorganisms can interfere positively in the
production of 3 and 4 in nymphs, evidencing a possible
symbiotic relationship.25
Compounds 4 and 5 showed very similar mass spectra,
but clearly different infrared spectra, as shown in Figure 3.
The IR spectrum for 4 (Figure 3a) showed two strong bands
at 1686 and 1674 cm-1, related to C=O stretching, with the
first one as typical from an α,β-unsaturated aldehyde and
the second as related to a ketone.26 Through co-injection
with the synthetic standard, the compound 4 was confirmed
as (E)-4-oxo-2-hexenal, a compound commonly found in
members of the Coreidae family.6,25
The lactone 5-ethyl-2(5H)-furanone was suggested
as being compound 5 through comparisons of its mass
spectra with the NIST database, in addition to infrared
Table 1. Relative abundance (mean standard deviation) of compounds present in Leptoglossus zonatus exuvial extracts from first to fifth-instars using
the Tukey’s test (p < 0.05)
Relative abundance / %
Compound
1st instar
0.6 0.1a
0.6 0.1a
42.9 3.7a
56.2 4.4a
Tr b
2nd instar
2.3 1.6b
1.2 0.9a
77.0 5.1b
14.6 5.0b
1.6 0.7ab
0.9 0.1a
3rd instar
1.3 0.2ab
3.3 5.8a
67.8 5.5b
25.3 7.4b
1.6 1.4ab
0.9 0.1a
4th instar
0.9 0.4ab
0.6 0.2a
71.4 7.2b
22.9 9.6b
2.2 1.7a
0.9 0.3a
5th instar
1.6 0.3ab
0.8 0.1a
(E)-3-Hexenal (1)
(Z)-2-Hexenal (2)
(E)-2-Hexenal (3)
72.1 7.03b
24.2 5.7b
0.7 0.5ab
0.5 0.4b
(E)-4-Oxo-2-hexenal (4)
5-Ethyl-2(5H)-furanone (5)
(E)-2-Octenal (6)
Tr c
Tr: relative abundance < 0.5 were not considered. Different letters on each line indicates statistical difference.

944
Identificaton of the Alarm and Sex Pheromones of the Leaf-Footed Bug, Leptoglossus zonatus
J. Braz. Chem. Soc.
Figure 3. Infrared (ZnSe, GC-FTIR) of compounds (a) 4 and (b) 5; and mass spectra (GC-MS) of (c) 4 and (d) 5 present in the dorsal abdominal glands
of Leptoglossus zonatus.
Scheme 1. Synthesis of 5-ethyl-2(5H)-furanone (5).
Table 2. Relative abundance (mean standard deviation) of the
compounds present in Leptoglossus zonatus MTG extracts using the
Tukey’s test (p < 0.05)
from hexanal and hexanoic acid (Figures S13 and S15a, SI
section) showed McLafferty rearrangements leading to the
base peak m/z 44 for 7 and 60 for 9. The structure of the
primary alcohol 8 was proposed due to the loss of water
with elimination of ethylene leading to the presence of the
Relative abundance / %
Compound
Male
Female
38.7 8.0b
5.1 1.1
base peak m/z 56 (Figure S14a, SI section). Esters 10 and 11
were suggested due to the fragment m/z 61, corresponding
to the rearrangement of two hydrogens leading to
[CH2C(OH)(OH2)]+ (Figures S16a and S17a, SI section).
GC-FTIR analyses were performed and spectra showed
different characteristic band absorptions of C=O stretching:
at 1720 cm-1 (aldehyde); at 1701 cm-1 (carboxylic acid);
and at 1740 cm-1 (ester) (Figures S13b, S15b, and S16b, SI
section). The broad and intense band at 3284 cm-1 indicated
an alcohol. The C–C–O asymmetric stretch of primary
alcohols appears at 1060 cm-1 (Figure S14b, SI section).
The most abundant compounds found in adults of
L. zonatus MTG are 7 and 10 (Table 2), with males
producing higher amounts of hexanal when compared with
Hexanal (7)
52.5 1.0a
3.1 2.7
1-Hexanol (8)
Hexanoic acid (9)
Hexyl acetate (10)
Octyl acetate (11)
6.7 10.4
36.9 13.9
0.9 0.3
13.2 3.0
41.8 5.7
1.2 0.3
Different letters on each line indicates statistical difference.
females. Leal et al.4 identified most of these compounds for
L. zonatus, except octyl acetate (11). However, the relative
abundance of the compounds differs from our data due to
the use of a different extraction methodology, immersing
the insects in hexane during 3 min.4,31 In this work, the
compounds were extracted directly from the pheromone-

Vol. 30, No. 5, 2019
Inoue et al.
945
Figure 4. GC analyses of aeration extracts from males and females Leptoglossus zonatus. Male specific compounds are highlighted.
producing glands. The components identified in L. zonatus
MTG are also found in other species of coreids as alarm
pheromone, not being specie-specific. In addition, the
components of the DAGs and the MTG are completely
different in Coreidae.6,24
reported to improve pheromonal attractiveness of species
of Coleoptera and Diptera.33,34
GC-EAD analysis of female antennae showed
active responses to five different compounds present in
L. zonatus extract from males (Figure 6). EAD active
compounds (13-17) were identified through GC-MS, GC-
FTIR, retention indexes and co-injection with authentic
standards. The mean amounts of compounds 13-17
emitted per insect in 24 h were 0.18 0.08 mg (12),
0.06 0.01 mg (13), 0.02 0.04 mg (14), 0.87 0.28 mg
(15) and 0.02 0.02 mg (16).
Quantification and production of sexual volatiles by
Leptoglossus zonatus
Analysis of aeration extracts from L. zonatus showed
nine male-specific compounds (Figure 4). Y-Olfactometer
bioassays (Figure 5), employing the extract from males,
suggested that these compounds are a sex pheromone of
this species, once that it was bioactive only to females
(p = 0.0012).
MS spectra of compounds 12 and 13 (Figures S18a and
S19a, SI section) suggested their identity as monoterpenes.
MS spectrum obtained from 12 suggested a 3,7-dimethyl-
1,3,6-octatriene structure. Retention index (1033) and
GC-FTIR spectrum (Figure S18b, SI section) allowed
the determination of the stereochemistry at C3 as cis,
suggesting 12 as (Z)-β-ocimene, with a fingerprint identical
to previously reported spectra.35 Band positions and
relative intensity profiles of the two C=C stretch bands in
the 1600 cm-1 region were useful for determination of the
configuration at C3 (1593 and 1647 cm-1), with the relative
intensity of the higher frequency band being weaker.
Out of phase =CH2 stretch (3091 cm-1), trans CH wag
(987 cm-1), and =CH wag (900 cm-1) bands, confirmed the
presence of the terminal double bond at C1. Compound 12
was confirmed as (Z)-β-ocimene after comparison with
analytical data obtained from a reference compound.
Mass spectrum (Figure S19a, SI section) and retention
index (1123) obtained for compound 13 suggested its
structure to be related to 2,6-dimethyl-2,4,6-octatriene.
IR spectrum showed a C–H stretch band at 3043 cm-1
confirming a structure with only internal double bonds
(Figure S19b, SI section). The C–H bending stretch
(1085 cm-1) suggested the stereochemistry at C4 and C6 as
cis-cis or trans-trans. Through comparison of previously
reported spectra, bands intensities at 1270 and 1238 cm-1
(on plane C–H stretches) confirmed the stereochemistry
Figure 5. Behavioral responses of females (n = 17) and males (n = 14)
Leptoglossus zonatus to aeration extracts from males in aY-olfactometer
using the binomial test (*p < 0.05).
The attractiveness of the male-specific pheromone
is observed when using the combination of extract from
males and parts of the host plants in the bioassays. The
volatiles of the host plant can enhance the attractiveness
of the sex pheromone because it indicates a good place for
oviposition.32 The presence of the host plant has also been

946
Identificaton of the Alarm and Sex Pheromones of the Leaf-Footed Bug, Leptoglossus zonatus
J. Braz. Chem. Soc.
Figure 6. Responses of female Leptoglossus zonatus antenna to the extract from males in GC-EAD analysis.
at C4 and C6 as cis-cis.36 Moreover, the retention index
of the isomer trans-trans is higher than the compound 13
(RItrans-trans: 1140; RI13: 1123). Compound 13 was identified
as Z,Z-allo-ocimene.
IR spectrum from 14 (RI: 1204) presented a strong
C=O stretching absorption at 1715 cm-1 and a C–H
stretching absorption at 2753 cm-1, typical for aldehydes
(Figure S20, SI section). Moreover, MS spectrum showed
typical fragments for long chain aldehydes, with base peak
at m/z 57, and the molecular ion at 156 m/z, suggesting its
structure as decanal.
The compounds 15 and 16 presented M+ at m/z 204, with
MS spectra related to sesquiterpenes. Comparison of these
compounds with the NIST mass spectral library suggested
α-trans-bergamotene (RI: 1438) to compound 15 and
(E)-β-farnesene (RI: 1458) to compound 16. IR spectrum
for compound 15 showed an absorption band in 3027 cm-1
(C–H stretch) (Figure S21b, SI section) revealing the
presence of internal trisubstituted double bonds. On the
other hand, IR spectrum obtained from 17 (Figure S22b,
SI section), suggested an (E)-β-type compound due to the
presence of the bands at 3089 cm-1, corresponding to out
of phase =CH2 stretch and at 1109 cm-1 (–C(CH3)=CH),
useful to determinate the stereochemistry at C3 as trans.
These compounds were identified through comparisons
of mass and infrared spectra and co-injection of isolated
reference compounds from essential oils of Copaifera sp.
and Matricaria recutita.37,38
A wide variety of structures are found as sex pheromones
in Pentatomidae, such as cis and trans-(Z)-α-bisabolene
epoxides in Nezara viridula and (R)-15-hexadecanolide
for Piezodorus hybneri.39,40 In Coreidae, the sex specific
compounds do not present structural patterns. Two specific
male compounds were identified in the ventral abdominal
gland of Pachylis laticornis as (E)-2-hexenyl tiglate and
(E)-2-hexenyl (E)-2-hexenoate.41 In Phthia picta the sex
pheromone is the hydrocarbon 5,9,17-trimethylhenicosane.42
Monoterpenes and sesquiterpenes were found
in male volatiles of Amblypelta lutescens lutescens
((–)-(3R)-(E)-nerolidol) and Leptoglossus phyllopus
((E)-nerolidol).9
Conclusions
In summary, the defensive compounds and the alarm
pheromone of L. zonatus were described. It is the first time
that 5-ethyl-2(5H)-furanone was identified as a defensive
compound of nymphs in this family. In addition, an evidence
for existence of male sex pheromone was found. Aeration
extracts from L. zonatus included five male-specific and
EAD active compounds that have high probability of
being related to the sex pheromone of the species. Further
investigation is underway in order to evaluate the effect of
these compounds in field experiments and the influence of
the host plant in the sexual communication of this species.

Vol. 30, No. 5, 2019
Inoue et al.
947
Supplementary Information
20. Mekonnen, A.; Yitayew, B.; Tesema, A.; Taddese, S.; Int. J.
Microbiol. 2016, 2016, 8.
Supplementary information (GC-MS, GC-FTIR, NMR)
are available free of charge at http://jbcs.sbq.org.br as PDF
file.
21. Tamura, R.; Chen,Y.; Shinozaki, M.;Arao, K.; Wang, L.; Tang,
W.; Hirano, S.; Ogura, H.; Mitsui, T.; Taketani, S.; Ando, M.;
Kataoka, T.; Bioorg. Med. Chem. Lett. 2012, 22, 207.
22. Minitab Inc.; Minitab 17 Statistical Software; State College,
PA, USA, 2010.
Acknowledgments
23. Ayres, M.; Ayres, M. J.; Ayres, D. L.; Santos, A. S.; BioEstat
5.0; Sociedade Civil Mamirauá: Belém, PA, Brazil, 2007.
24. Prudic, K. L.; Noge, K.; Becerra, J. X.; J. Chem. Ecol. 2008,
34, 734.
The authors would like to thank Dr Daiane Szczerbowski
and MSc Samara Mendes for contributions to this
manuscript and Dr Rodolfo Bianco for capture and donation
of insects. This reasearch was supported by INCT
Semioquímicos na Agricultura, CNPq and CAPES.
25. Becerra, J. X.; Venable, G. X.; Saeidi, V.; J. Chem. Ecol. 2015,
41, 593.
26. Silverstein, R. M.; Webster, F. X.; Kiemle, D. J.; Spectrometric
Identification of Organic Compounds, 7th
References
ed.; John Wiley &
Sons: Hoboken, 2005.
1. Xiao,Y. F.; Fadamiro, H.Y.; J. Appl. Entomol. 2010, 134, 694.
2. Matrangolo, W.; Waquil, J.; An. Soc. Entomol. Bras. 1994, 23,
419.
27. Cai, L.; Koziel, J.; O’Neal, M.; Chromatography 2015, 2, 265.
28. Farneti, B.; Alarcón, A. A.; Papasotiriou, F. G.; Samudrala, D.;
Cristescu, S. M.; Costa, G.; Harren, F. J. M.; Woltering, E. J.;
Food Bioprocess Technol. 2015, 8, 1442.
3. Marchiori, C. H.; Cienc. Rural 2003, 33, 767.
4. Leal, W. S.; Panizzi, A. R.; Niva, C. C.; J. Chem. Ecol. 1994,
20, 1209.
29. Farine, J. P.; Everaerts, C.; Brossut, R.; Le Quére, J. L.; Biochem.
Syst. Ecol. 1993, 21, 363.
5. Aldrich, J. R.; Annu. Rev. Entomol. 1988, 33, 211.
6. Noge, K.; Kakuda, T.; Abe, M.; Tamogami, S.; J. Chem. Ecol.
2015, 41, 757.
30. Solomon, D.; Dutcher, D.; Raymond, T.; J. Air Waste Manage.
Assoc. 2013, 63, 1264.
31. Zarbin, P. H. G.; Ferreira, J. T. B.; Leal, W. S.; Quim. Nova
1999, 22, 263.
7. Millar, J. G.; Top. Curr. Chem. 2005, 240, 37.
8. Fountain, M.; Jåstad, G.; Hall, D.; Douglas, P.; Farman, D.;
Cross, J.; J. Chem. Ecol. 2014, 40, 71.
32. Blatt, S. E.; Borden, J. H.; Can. Entomol. 1996, 128, 777.
33. Yasui, H.; Fujiwara-Tsujii, N.; Sci. Rep. 2016, 6, 29526.
34. Bachmann, G. E.; Segura, D. F.; Devescovi, F.; Juárez, M. L.;
Ruiz, M. J.;Vera, M. T.; Cladera, J. L.; Teal, P. E.A.; Fernández,
P. C.; PLoS One 2015, 10, e0124250.
9. Aldrich, J. R.; Waite, G. K.; Moore, C.; Payne, J. A.; Lusby, W.
R.; Kochansky, J. P.; J. Chem. Ecol. 1993, 19, 2767.
10. Khrimian, A.; Fay, H. A. C.; Guzman, F.; Chauhan, K.; Moore,
C.; Aldrich, J. R.; Psyche 2012, ID 536149.
35. Svatoš, A.; Attygalle, A. B.; Anal. Chem. 1997, 69, 1827.
36. Mitzner, B. M.; Theimer, E. T.; Freeman, S. K.; Appl. Spectrosc.
1965, 19, 169.
11. Paul, K. C.; Sinsheimer, J. S.; Cockburn, M.; Bronstein, J. M.;
Bordelon, Y.; Ritz, B.; Environ. Int. 2017, 107, 75.
12. Gallo, D.; Nakano, O.; Wiendel, F. M.; Silveira Neto, S.;
Carvalho, R. P. L.; de Batista, G. C.; Berti Filho, E.; Para, J.
R. P.; Zucchi, R. A.; Alves, S. B.; Vendramin, J. D.; Manual de
Entomologia Agrícola; FEALQ: Piracicaba, 2002.
13. Stenberg, J. A.; Trends Plant Sci. 2017, 22, 759.
14. Raguso, R. A.; Agrawal, A. A.; Douglas, A. E.; Jander, G.;
Kessler, A.; Poveda, K.; Thaler, J. S.; Ecology 2015, 96, 617.
15. Witzgall, P.; Kirsch, P.; Cork, A.; J. Chem. Ecol. 2010, 36, 80.
16. Borges, M.; Aldrich, J. R.; Experientia 1992, 48, 893.
17. Fávaro, C. F.; Santos, T. B.; Zarbin, P. H. G.; J. Chem. Ecol.
2012, 38, 1124.
37. Ziech, R. E.; Farias, L. D.; Balzan, C.; Ziech, M. F.; Heinzmann,
B. M.; Lámeira, O. A.; de Vargas, A. C.; Pesq. Vet. Bras. 2013,
33, 909.
38. Heuskin, S.; Godin, B.; Leroy, P.; Capella, Q.; Wathelet, J. P.;
Verheggen, F.; Haubruge, E.; Lognay, G.; J. Chromatogr. A
2009, 1216, 2768.
39. Brézot, P.; Malosse, C.; Mori, K.; Renou, M.; J. Chem. Ecol.
1994, 20, 3133.
40. Leal, W. S.; Kuwahara, S.; Ono, M.; Meinwald, J.; J. Chem.
Ecol. 1998, 24, 1817.
41. Aldrich, J. R.; Kochansky, J. P.; Lusby, W. R.; Dutky, S. R.; J.
Chem. Ecol. 1982, 8, 1369.
18. Feldlaufer, M. F.; Domingue, M. J.; Chauhan, K. R.; Aldrich,
J. R.; J. Med. Entomol. 2010, 47, 140.
42. Soldi, R. A.; Rodrigues, M. A. C. M.; Aldrich, J. R.; Zarbin, P.
H. G.; J. Chem. Ecol. 2012, 38, 814.
19. Bardají, D. K. R.; da Silva, J. J. M.; Bianchi, T. C.; Eugênio,
D. S.; de Oliveira, P. F.; Leandro, L. F.; Rogez, H. L. G.;
Venezianni, R. C. S.; Ambrosio, S. R.; Tavares, D. C.; Bastos,
J. K.; Martins, C. H. G.; Anaerobe 2016, 40, 18.
Submitted: May 9, 2018
Pubished online: December 10, 2018
This is an open-access article distributed under the terms of the Creative Commons Attribution License.